The long-term goal of our research is to understand how microtubule arrays are created, maintained and remodeled to enable cells to perform different functions, change shape and adapt to changing conditions. Regulation of polymer number, length and turnover lies at the heart of this problem and the microtubule severing protein katanin has emerged as a key protein for this task as evidenced by its important role in neuronal development, meiotic and mitotic spindle assembly and function, cell migration, ciliary assembly and disassembly, and cell morphogenesis. However, we have very limited knowledge of the mechanisms controlling the location, timing and outcome of katanin activity. To address this shortcoming, we propose the following three specific aims: (1) What are the mechanisms for targeting katanin to specific microtubule sites? We hypothesize that the p80 regulatory subunit targets the p60 catalytic subunit of katanin by preferentially binding to intersecting microtubule geometries and that off-target p60 katanin binding is inhibited by microtubule-associated proteins. To test this hypothesis, we will use a combination of in vitro reconstitution, cryo-EM and cellular studies to: (i) determine the necessary and sufficient conditions to recruit p60 katanin to microtubule crossover sites; (ii) elucidate the structure and conformation of p60 katanin, with and without p80, during microtubule severing; and (iii) determine how the MAP65 protein protects microtubules against katanin activity. (2) What are the mechanisms regulating p60 katanin severing activity? We hypothesize that p60 katanin activity is held tightly in check by multiple regulatory mechanisms to provide nuanced control depending on the cellular context. To test this hypothesis, we will focus on two newly identified regulatory proteins, RIC1 and SPR2, which are proposed to control the rate and location of p60 activity respectively. We will use in vitro reconstitution and live imaging to examine how RIC1 boosts the severing activity of p60 katanin, and how SPR2 regulates access of p60 katanin to microtubule crossover sites. (3) What are the mechanisms regulating the dynamics of severed microtubule ends? We hypothesize that microtubule plus-end tracking proteins play a major role in determining the outcome of microtubule severing by regulating the dynamics of the new plus-ends. To test this hypothesis, we will focus on a +TIP called SPR1, which is implicated as a rescue factor for severed microtubules by our preliminary data. We will use in vitro reconstitution with dynamic microtubules to determine whether and how SPR1 promotes rescue of microtubule plus ends created by severing; and use live cell experiments to test whether microtubule amplification via severing drives light-induced array reorientation and whether cells regulate the level of SPR1 via proteolysis to modulate the extent of rescue of severed microtubules.